Polysaccharides from prasinococcales

ABSTRACT

There is provided a composition comprising a polysaccharide obtainable from the microalgae,  Prasinococcus capsulatus  and strains related to  P. capsulatus  for use prophylactically and/or therapeutically in the treatment of disorders of the immune system, for example in psoriasis and dermatological conditions, internal immune system disorders, in particular gut inflammatory conditions and respiratory conditions. Further, there is provided derivatives of a polysaccharide obtainable from the microalgae,  Prasinococcus capsulatus  or an algal strain related to  P. capsulatus  and the use of such derivatives prophylactically and/or therapeutically in the treatment of disorders of the immune system.

FIELD OF THE INVENTION

The invention relates to a composition comprising a polysaccharideobtainable from the microalgae, Prasinococcus capsulatus and strainsrelated to P. capsulatus for use prophylactically and/or therapeuticallyin the treatment of disorders of the immune system, such as inflammatorydisorders, for example in psoriasis and dermatological conditions.Further, there is provided derivatives of a polysaccharide obtainablefrom the microalgae, Prasinococcus capsulatus or an algal strain relatedto P. capsulatus and the use of such derivatives prophylactically and/ortherapeutically in the treatment of disorders of the immune system, suchas inflammatory disorders, for example in psoriasis and dermatologicalconditions.

Also provided is the use of polysaccharide and derivatives thereofobtainable from the microalgae, Prasinococcus capsulatus or an algalstrain related to P. capsulatus in the preparation of cosmetic andnutritional compositions.

BACKGROUND OF THE INVENTION

Macroalgae (seaweeds) have been exploited to provide long establishedproducts such as alginate and carageenan and newer products such asfucoidan; however, microalgae have not yet been significantly used inthis way and there has been relatively little characterisation of thoseproducts derived from microalgae.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a gelforming polysaccharide obtainable from Prasinococcus capsulatus or analgal strain related to P. capsulatus wherein the gel formingpolysaccharide is a sulphated heteropolymer of molecular weight greaterthan 670 kDa comprising glucose, galactose, arabinose and uronic acidunits for use in the treatment of immune system disorders, in particularimmune system disorders which are inflammatory conditions.

The inventors have undertaken a range of methods to determine thecomposition of the polysaccharide of the invention and the derivativesthereof. Using the methods detailed herein, without wishing to be boundby theory as to the configuration of the polysaccharide, the inventorshave characterised the polysaccharide and derivatives as described.Therefore, in embodiments when a polysaccharide or derivatives arecharacterised using the methods described herein they provide themonosaccharide and sulphate compositions as described herein.

Prasinococcus capsulatus, a relatively recently discovered species hasbeen shown to produce polysaccharides (Miyashita, et al. Prasinococcuscapsulatus Gen. Et Sp. Nov., A New Marine Coccoid Prasinophyte. J. Gen.Appl. Microbiol., 39, 571-582 (1993) and Miyashita, et al. Compositionand nature of extracellular polysaccharide produced by newly isolatedcoccoid prasinophyte, Prasinococcus capsulatus. J. Marine Biotechnol.,3, 136-139 (1995).).

Suitably a strain related to Prasinococcus capsulatus may include astrain of the order Prasinococcales. In embodiments a strain related toPrasinococcus capsulatus can include Prasinoderma singularis. Inembodiments the polysaccharide can be polysaccharide associated with thecell wall of the microalgae, and/or be present in a homengenate of themicroalgae, and/or secreted polysaccharide or exopolysaccharide. Thepolysaccharide may be provided in an isolated, purified, orsemi-purified form. In embodiments the polysaccharides can be a purifiedmaterial that has been separated from cell biomass, such that thepolysaccharide is at least 50% polysaccharide by weight, and morepreferably above 75% polysaccharide by weight, more preferably above 85%by weight, more preferably about 95% by weight.

In embodiments, immune system disorders are those where the response ofvascular cells and tissues to internal or external stimuli isinsufficient, excessive or chronic. In inflammatory conditions thisresponse is generally excessive and/or chronic resulting in increasedand maintained activation of immune cells (such as neutrophils andT-cells), which may infiltrate tissues and increase production ofpro-inflammatory mediators, resulting in sustained inflammation. It hasbeen determined that polysaccharides can be used to moderate the effectsof this activation, for example, by reducing the activity of neutrophilproteases, such as elastase; by reducing the secretion ofpro-inflammatory proteins (cytokines) and reactive oxygen species fromblood and endothelial cells; and by reducing blood cell infiltration toeffected tissues.

In embodiments of the invention, a polysaccharide as described hereincan be for use in the treatment of inflammatory skin conditions,including eczema, psoriasis and atopic dermatitis.

In embodiments of the invention a polysaccharide as described herein canbe for use in the treatment of inflammatory conditions of the gut, inparticular for use in the treatment of bowel disorders, includingirritable bowel syndrome, Crohn's disease and ulcerative colitis.

In embodiments of the invention a polysaccharide as described herein canbe for use in the treatment of respiratory inflammatory conditionsincluding asthma, cystic fibrosis, emphysema, chronic obstructivepulmonary disorder, acute respiratory distress syndrome, or allergicrhinitis.

Embodiments of the polysaccharide for use in the invention can becharacterised by at least one, at least two, at least three, at leastfour, or at least five of the characteristics (i), (ii), (iii), (iv),and (v).

(i) Molecular weight range

(ii) Monosaccharide composition

(iii) Immunomodulatory activity

(iv) Sulphate content (as a percentage of molecular weight of themolecule)

(v) Viscosity/gel-forming properties

Molecular Weight Range

A polysaccharide for use in the present invention typically has amolecular weight of 670 kDa or greater than 670 kDa.

In embodiments the polysaccharide of the invention can have a molecularweight in the range of 670 kDa to 40 MegaDa. In embodiments, thepolysaccharide of the invention can have a molecular weight greater thanor equal to 1 MegaDa, greater than or equal to SMegaDa, greater than orequal to 10 MegaDa, greater than or equal to 15 MegaDa, greater than orequal to 20 MegaDa, greater than or equal to 25 MegaDa, greater than orequal to 30 MegaDa, greater than or equal to 35 MegaDa, or 40 MegaDa.

In embodiments a polysaccharide of the present invention can compriseabout

20 to 30% Glucose

30 to 60% Galactose

4 to 19% Arabinose

2 to 6% Uronic acids

and a small percentage (1 to 10%) of other sugars, more particularly

1-4% Rhamnose

1-3% Xylose

1-10% Mannose

(% by weight).

In embodiments a polysaccharide of the present invention can compriseabout

25 to 29% Glucose

35 to 47% Galactose

14 to 15% Arabinose

4 to 6% Uronic acids

and a small percentage (1 to 4%) of other sugars, more particularly

2.4% Rhamnose

1.8% Xylose

3.3% Mannose

(% by weight).

In embodiments a polysaccharide for use in the present invention canhave a sulphate content of about 17 to 35% by weight, 17 to 30% byweight, suitably, 25 to 30% by weight, suitably 17 to 25% by weight,suitably the polysaccharide may have a sulphate content of about 20% byweight, suitably about 19% by weight.

In embodiments a polysaccharide for use in the invention can demonstrateimmunomodulatory activity in vitro, wherein for example thepolysaccharide can inhibit neutrophil elastase activity by about 60 to90%, in particular 60 to 80% relative to neutrophils to which thepolysaccharide is not provided.

As well as being useful therapeutically to treat inflammatory conditionsof the skin, it is considered embodiments of the polysaccharidesdescribed herein can be used to treat internal immune system disorders,in particular gut inflammatory conditions and respiratory conditions,for example gut inflammatory conditions including bowel disorders,irritable bowel syndrome, Crohn's disease, and ulcerative colitis andrespiratory conditions including asthma, cystic fibrosis, emphysema,chronic obstructive pulmonary disorder, acute respiratory distresssyndrome, or allergic rhinitis. Treatment of such conditions can be viaingestion of a polysaccharide or by inhalation of the polysaccharide. Itis further considered that where a patient has an underlying immunesystem disorder, but no symptoms are present, then a polysaccharide asdescribed herein may be provided to minimise the risk of symptoms.

Accordingly a second aspect of the present invention provides anutritional supplement comprising a polysaccharide obtainable from P.capsulatus or a strain related to P. capsulatus wherein the gel formingpolysaccharide is a sulphated heteropolymer, of molecular weight greaterthan 670 kDa, primarily comprising glucose, galactose, arabinose anduronic acid units.

Additionally, where no therapeutic benefit is required, the inventorsconsider that the polysaccharide may usefully provide cosmeticadvantages for users.

Accordingly a third aspect of the invention is a cosmetic preparationcomprising a polysaccharide obtainable from Prasinococcus capsulatus ora strain related to P. capsulatus wherein the gel forming polysaccharideis a sulphated heteropolymer of molecular weight greater than 670 kDaprimarily comprising glucose, galactose, arabinose and uronic acidunits.

As will be appreciated, the embodiments of the polysaccharide of thefirst aspect of the invention can be used in the second and thirdaspects of the invention.

Derivatives of Polysaccharide and their Use

The inventors have determined that derivatives of a gel formingpolysaccharide obtainable from Prasinococcus capsulatus or a strainrelated to P. capsulatus wherein the gel forming polysaccharide is asulphated heteropolymer of molecular weight greater than 670 kDacomprising glucose, galactose, arabinose and uronic acid units can alsobe used in the treatment of immune system disorders, in particularimmune system disorders which promote an inflammatory response.

Accordingly a fourth aspect of the invention provides a derivative(oligosaccharide) of a gel forming polysaccharide from Prasinococcuscapsulatus or a strain related to P. capsulatus wherein the gel formingpolysaccharide is a sulphated heteropolymer of molecular weight greaterthan 670 kDa primarily comprising glucose, galactose, arabinose anduronic acid units wherein said derivative has a molecular weight in therange of about 2 kDa to 10 kDa, or in the range of about 2 to 20 kDa, orin the range of about 20 to 60 kDa.

As will be appreciated, embodiments of the polysaccharide of the firstaspect of the invention can be used to form derivatives of the fourthaspect of the invention.

In embodiments a derivative can be a low molecular weight fragment witha molecular weight in the range 2 to 10 kDa. In embodiments a derivativecan comprise a larger fragment of polysaccharide with a molecular weightin the range 20 to 60 kDa.

In embodiments low molecular weight fragments of the polysaccharide ofaround 2 to 10 kDa can comprise about

30 to 40% Glucose

30 to 40% Galactose

8 to 14% Arabinose

7 to 11% Uronic acid

and a small percentage (1 to 10%), suitably a small percentage (1 to 4%)of other sugar units (% by weight).

In embodiments low molecular weight fragments of the polysaccharide ofaround 2 to 10 kDa can comprise about

35% Glucose

35% Galactose

11% Arabinose

9% Uronic acid

and a small percentage (1 to 10%), suitably a small percentage (1 to 4%)of other sugar units (% by weight).

In embodiments a large fragment of the polysaccharide (around 20 to 60kDa) can comprise

25 to 28% Glucose

35 to 55%, suitably 35 to 45% Galactose

8 to 17% Arabinose, suitably 15% Arabinose

4-6% Uronic acids

and a small percentage (1 to 4%) of other sugar units (% by weight).

In embodiments a large fragment of the polysaccharide (around 20 to 60kDa) can comprise

25% Glucose

50% Galactose

15% Arabinose

5% Uronic acids

and a small percentage (1 to 5%) of other sugar units (% by weight).

In embodiments a derivative of a polysaccharide of the invention cancomprise about

8.4% Arabinose

0.7% Rhamnose

2.0% Xylose

2.2% GalA (galacturonic acid)

57.9% Galactose

24.6% Glucose

3.8% GlcA (glucuronic acid)

by weight.

Suitably, in embodiments, a derivative of a polysaccharide of thepresent invention can have a sulphate content of about 20 to 30% byweight, suitably 25 to 30% by weight.

In embodiments an oligosaccharide derivative of the polysaccharide caninhibit neutrophil elastase release by about 70 to 90%, suitably 80 to90% relative to neutrophils to which derivative is not provided

In embodiments a derivative of the polysaccharide can inhibit neutrophilreactive oxygen species (ROS) production by about 30-40% relative toneutrophils in which derivative is not provided. In embodiments anoligosaccharide derivative can inhibit human keratinocyte IL-8 geneexpression and release by about 70 to 100% relative to keratinocytes towhich derivative is not provided. This activity compares to thewell-characterised polysaccharide anticoagulant drug heparin, whichinhibits elastase release by about 60-75% but which has no significanteffect on ROS production. The activity also compares to the seaweedpolysaccharide fucoidan, which inhibits elastase release by about 70 to90%, and keratinocyte IL-8 release by about 80 to 90%.

In embodiments an oligosaccharide derivative of the polysaccharide caninhibit human keratinocyte IL6 and IL17C release by about 50-70%relative to human keratinocytes to which derivative is not provided. Inembodiments an oligosaccharide derivative of the polysaccharide caninhibit the release of interferon gamma from human peripheral bloodmononuclear cells (PBMCs) by about 50-70% relative to human PBMC's inwhich derivative is not provided. In embodiments an oligosaccharide caninhibit the chemotaxis of human neutrophils by about 50-70% and of THP-1(monocyte) cells by about 30-50% relative to neutrophils and monocytesto which the derivative is not provided. In embodiments anoligosaccharide derivative of the polysaccharide can inhibit imiquimodinduced mouse skin inflammation in a dose dependent manner relative tocontrols where the derivative is not provided.

By about is meant within 1 to 20%, more particularly within 10%, yetmore particularly within 5%, even yet more particularly within 2% of thestated value.

Embodiments of derivatives of the invention can be for use in thetreatment of immune system disorders, in particular immune systemdisorders which promote an inflammatory response, more specifically skinconditions, including eczema, psoriasis and atopic dermatitis.

Embodiments of derivatives of the invention can be for use in thetreatment of internal immune system disorders, in particular gutinflammatory conditions and respiratory conditions, for example gutinflammatory conditions including bowel disorders, irritable bowelsyndrome, Crohn's disease, and ulcerative colitis and respiratoryconditions including asthma, cystic fibrosis, emphysema, chronicobstructive pulmonary disorder, acute respiratory distress syndrome, orallergic rhinitis.

The use of such derivatives in such treatments provides a further aspectof the invention.

Suitably embodiments of derivatives may be prepared by any method knownin the art, including hydrolysis or enzymatic hydrolysis thepolysaccharides or by free radical or photochemical methods, such asthat described by Higashi et al

(Controlled photochemical depolymerization of K5 heparosan, abioengineered heparin precursor, Carbohydrate Polymers 86 (2011)1365-1370).

Embodiments of derivatives of the invention can be depolymerisedpolysaccharides prepared by a free radical or photochemical method.

Embodiments of the derivatives can be oligosaccharides with a sulphatecontent of about 25-30% by weight.

Preferably an oligosaccharide derivative of the invention can haveimmunomodulatory properties equivalent or greater than the nativepolysaccharide material of the invention.

According to a fifth aspect of the invention there is provided acosmetic preparation comprising at least one derivative of apolysaccharide from Prasinococcus capsulatus or a strain related to P.capsulatus wherein the gel forming polysaccharide is a sulphatedheteropolymer of molecular weight greater than 670 kDa primarilycomprising glucose, galactose, arabinose and uronic acid units whereinsaid derivative has a molecular weight in the range 2 kDa to 10 kDa orin the range of about 20 to 60 kDa.

In embodiments a derivative can be a low molecular weight fragment witha molecular weight in the range 2 to 10 kDa. In embodiments a derivativecan comprise a large fragment of polysaccharide with a molecular weightin the range 20 to 60 kDa.

In embodiments, a combination of a large fragment of a derivative andlow molecular weight fragment may be provided.

According to a sixth aspect of the invention there is provided anutritional supplement comprising at least one derivative of apolysaccharide from Prasinococcus capsulatus or a strain related to P.capsulatus wherein the gel forming polysaccharide is a sulphatedheteropolymer of molecular weight greater than 670 kDa primarilycomprising glucose, galactose, arabinose and uronic acid units whereinsaid derivatives have a molecular weight in the range 2 kDa to 10 kDa orin the range of about 20 to 60 kDa.

In embodiments a derivative can be a low molecular weight fragment witha molecular weight in the range 2 to 10 kDa. In embodiments a derivativecan comprise a large fragment of the polysaccharide with a molecularweight in the range 20 to 60 kDa.

In embodiments, a combination of a large fragment of a derivative andlow molecular weight fragment may be provided.

Compositions

Suitably a polysaccharide or derivative as discussed herein can beprovided as part of a composition. Such a composition may be suitablefor oral, topical, rectal or parenteral, nasal or pulmonaryadministration (by inhalation). In embodiments the composition can beeither for topical application to the skin or for ingestion according touse.

In embodiments a ready-for-use composition can be in the form of atablet, capsule, cachet, or as a dispersible granule, which may be, forexample, suspended in water before administration or sprinkled on food.A composition may conveniently be presented in unit dosage form and maybe prepared by any of the methods well-known in the food industry forthe preparation of food and food supplements, or by methods known to thepharmaceutical industry for use as a pharmaceutical, for example as atopical medication.

Compositions for topical administration may be provided, for example, asa gel, cream or ointment. Such compositions can be applied directly tothe skin or carried on a suitable support, such as a bandage, gauze,mesh or the like that can be applied to an area to be treated.

Methods known to those skilled in the art of food manufacturing include,but are not limited to; dry-blending of active agents and otheringredients in powder form, spray-drying of emulsions containing allcomponents or the use of extrusion technologies to form pellets orgranules. Alternatively, the composition may be the form of a liquidtonic.

A polysaccharide or derivative may be provided as a pharmaceuticallyacceptable salt or pharmaceutically acceptable solvate. In embodimentsthe polysaccharide or derivative can be administered alone, or inadmixture with a pharmaceutical carrier, excipient or diluent selectedwith regard to the intended route of administration and standardpharmaceutical practice. A pharmaceutical carrier can be aphysiologically acceptable carrier, either organic or inorganic, naturalor synthetic with which the polysaccharide or derivative thereof of thepresent invention can be combined to facilitate the application.

In embodiments a polysaccharide or derivative can be admixed with anysuitable binder (s), lubricant (s), suspending agent (s), coating agent(s), solubilising agent (s), carrier(s), or buffer stabiliser(s).

A composition of the invention may also contain one or more furtheractive compounds selected as necessary for the condition being treated.For example a composition may comprise a further active compound whichtargets distinct pathways or mechanisms from that targeted by theproduct of the invention. This may provide improved efficacy, forexample a synergistic effect. In embodiments the polysaccharide orderivative can be provided in combination with Vitamin D.

Cosmeceutical or Cosmetic Preparation

In embodiments a polysaccharide or derivative of the present inventioncan be provided as a cosmeceutical, i.e. a cosmetic product withbiologically active ingredients purporting to have medical or drug likebenefits.

Alternatively, a polysaccharide or derivative of the present inventioncan be provided as a cosmetic which improves the appearance and functionof the skin, but does not have a clinical effect.

Suitably, compositions for use as a cosmeceutical or cosmeticpreparation can be provided as known in the art, including, but notlimited to skin creams, gels, serums, washes, rinses, shampoos,conditioners, mousses and the like.

Embodiments of a cosmetic preparation of the invention can be providedfor example as a cream, serum, gel or ointment for topicaladministration to the skin. In embodiments the cosmetic preparation canbe for use as an anti-aging skin preparation, for use in skintoning/smoothing or to alter the colour of the skin. Such preparationsmay suitably minimise effects considered to be related to aging such asthe visual appearance of wrinkles, sun-damage and may increase skinelasticity. In such cosmetic compositions the polysaccharide orderivative thereof can typically be provided in combination with a basecarrier or skin moisturising substance. Suitably a base carrier iscompatible with the other ingredients of the composition and notdeleterious to the user of the cosmetic. Typically such preparations mayfurther include preservatives, fragrance or anti-oxidants. Additionally,such preparations may include water, wetting agents, alcohols, oils,colourants and the like.

In embodiments a cosmetic preparation can be provided comprising apolysaccharide for use in the invention. In preferred embodiments acosmetic preparation can be provided with a derivative as discussedherein.

Nutritional Supplement

In embodiments a nutritional supplement of the invention can promote ahealthy gut in the subject which receives the nutritional supplement.Suitably embodiments of a nutritional supplement can decrease crampingor discomfort in the bowel. In embodiments a nutritional supplement canbe provided comprising a polysaccharide for use in the invention. Inpreferred embodiments a nutritional supplement can be provided with aderivative as discussed herein.

In embodiments a nutritional supplement of the invention can beformulated in capsule form to be taken orally. In embodiments anutritional supplement can be provided as part of a neutraceuticalcomposition.

Preparation of a Polysaccharide

In embodiments, a polysaccharide for use in the invention or derivativesof such a polysaccharide can be a polysaccharide isolated from thecellular or secreted fraction of a culture of Prasinococcus capsulatusor a strain related to P. capsulatus.

In embodiments the polysaccharide for use in the invention or at leastone derivative of such a polysaccharide can be isolated from thecellular fraction of a culture of Prasinococcus capsulatus or a strainrelated to P. capsulatus.

In embodiments the polysaccharide for use in the invention or at leastone derivative of such a polysaccharide can be isolated from thesecreted fraction of a culture of Prasinococcus capsulatus or a strainrelated to P. capsulatus.

In embodiments, a culture of Prasinococcus capsulatus can be thePrasinococcus capsulatus algal strain CCMPII94. This algal strain ispublicly available. The culture can be suitably grown in algal culturemedia as would be known in the art wherein modifications of thenitrogen, vitamin, silica or trace metals provided in the algal mediamay be made as would be known to one of skill in the art. The algalculture medium can be used with a sea water base or using synthetic seawater.

In embodiments a f/2 growth medium can be used with the followingcomposition:

Stock Solutions:

Trace elements: g/Liter Vitamin mix: g/Liter Na₂EDTA•2H₂O 4.16 VitaminB₁₂ (cyanocobalamin] 0.0005 FeCl₃•6H₂O 3.15 Thiamine HCl (Vitamin B₁)0.1 CuSO₄•5H₂O 0.01 Biotin 0.0005 ZnSO₄•7H₂O 0.022 CoCl₂•6H₂O 0.01MnCl₂•4H₂O 0.18 Na₂MoO₄•2H₂O 0.006

Such culture media is discussed by Sieburth, et al. Widespreadoccurrence of the oceanic ultraplankter, Prasinococcus capsulatus(prasinophyceae), the diagnostic “golgi-decapore complex” and the newlydescribed polysaccharide “capsulan”. J. Phycol. 35, 1032-1043 (1999) andGuillard R. and Ryther J. 1962 Studies of marine planktonic diatoms.Can. J. Microbiol. 8: 229-239.

Suitable medium can be made by adding the following to 950 mls filteredsea water (salinity 29-32 ppt).

Medium:

g/Liter NaNO₃ 0.075 NaH₂PO₄•2H₂O 0.00565 Trace element stock solution 1mL Vitamin mix stock solution 1 mL

Typically the pH can be set to 8.0 (2 ml 1M Tris-HCl at pH8 per liter ofmedium) and media made up to 1 liter with sea water. Medium can besterilised by autoclaving (eg. 121° C., 15 mins) and stored at 2-8° C.

Variations for P. capsulatus culture can be used wherein 80% ofNaH₂PO₄.2H₂O as above is used, plus 100 mg sodium glycerophosphate/literand wherein the twice the concentration of NaNO₃ and vitamins isincluded to increase biomass.

According to a further aspect of the present invention, there isprovided a method to produce the polysaccharide for use in the inventionor a derivative of the polysaccharide wherein the method comprises thesteps:

culture of microalgae, suitably a culture of Prasinococcus capsulatus,in particular the Prasinococcus capsulatus algal strain CCMPII94,

separation of microalgal biomass from culture medium,

concentration and desalting of the culture medium and

drying of the culture media.

Suitably, separation of the microalgae biomass from the medium may be bycentrifugation. In alternative embodiments separation may suitably beperformed by filtration, flocculation, or tangential flow filtration.Suitably concentration may be by tangential flow filtration. Inembodiments this may be by using a 100 kDa membrane. This may allowdesalting of the medium if diafiltration is also carried out.

Suitably, separation of the polysaccharide from the medium may beprovided by precipitation,

dialysis,

tangential flow filtration and/or

ion exchange chromatography

In embodiments, separation is provided by ion exchange chromatographyand concentration by tangential flow filtration.

After separation the media fraction may be dried. Drying may beperformed using, for example, lyophilisation and heat drying, shelfdrying using reduced atmospheric pressure or vacuum to dry at roomtemperature (20 degrees C.), spray drying, rotary drying, or spin flashdrying.

Suitably drying may be by spray drying of concentrated and desaltedmedium.

The cells (cell pellets) can also be processed to extract the targetpolysaccharide, for example the step of extracting may be a step of hotwater extraction or an enzymatic digest step or another suitableextraction protocol.

Extraction may be performed using for example, pressure disruption, ballmilling, sonication, or blending (high speed or Waring).

In preferred embodiments the method can provide a derivative(s) of thepolysaccharide wherein the derivatives are prepared by depolymerisationof the native polysaccharide by a free-radical or photochemical method,followed by fractionation using size exclusion chromatography ortangential flow chromatography to produce oligosaccharide fractions ofdefined molecular weight.

Treatment

A polysaccharide, a derivative thereof or a composition containing thepolysaccharide or a derivative thereof may be used to treat a number ofmedical conditions. Treatment includes any regime that can benefit ahuman or non-human animal. The treatment may be in respect of anexisting condition or may be prophylactic (preventative treatment).Treatment may include curative, alleviation or prophylactic effects.

Suitably a polysaccharide, a derivative thereof or a compositioncontaining the polysaccharide or a derivative thereof may be provided asa tablet or capsule. Suitably, a polysaccharide, a derivative thereof ora composition containing the polysaccharide or a derivative thereof maybe administered in a sustained release formulation. Suitably thepolysaccharide, a derivative thereof or a composition containing thepolysaccharide or a derivative thereof can be provided as a dietarysupplement to an animal, including humans, that will provide aprotective benefit to the animal and/or to be used therapeutically tomodulate the immune response, in particular to modulate the inflammatoryresponse, of the animal in particular a human.

Administration

The invention provides a polysaccharide for use or a derivative of thepresent invention, or a composition containing the same, for use as amedicament. The medicament may be for human usage or veterinary usage.Suitably in veterinary usage the animal patient may be a terrestrialanimal, more suitably a companion or performance animal. Suitably apatient may be a human. Suitably, a derivative of the polysaccharide ora composition containing the polysaccharide or a derivative thereof canbe applied topically to the patient, e.g. applied to the skin.

The product of the present invention may be administered by oral,topical, rectal or parenteral, nasal or pulmonary (by inhalation)routes. Typically, the physician will determine the actual dosage whichwill be most suitable for an individual patient and it will vary withthe age, weight and response of the particular patient. In general, atherapeutically effective daily oral dose of the product of theinvention is likely to range from 1 to 50 mg/kg body weight of thesubject to be treated, preferably 10 to 20 mg/kg. The above dosages areexemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

Accordingly, there is provided a method of administration of thepolysaccharide of the first aspect of the invention or a derivative ofthe fourth aspect of the invention for the treatment of disorders of theimmune system, in particular inflammatory conditions, more particularlyinflammatory conditions of the skin, including eczema, psoriasis andatopic dermatitis and/or internal immune system disorders, in particulargut inflammatory conditions and respiratory conditions, for example gutinflammatory conditions including bowel disorders, irritable bowelsyndrome, Crohn's disease, and ulcerative colitis and respiratoryconditions including asthma, cystic fibrosis, emphysema, chronicobstructive pulmonary disorder, acute respiratory distress syndrome, orallergic rhinitis wherein the method comprises providing atherapeutically effective amount of the polysaccharide and/or derivativeto a subject in need thereof.

Preferred features and embodiments of each aspect of the invention areas for each of the other aspects mutatis mutandis unless context demandsotherwise.

Each document, reference, patent application or patent cited in thistext is expressly incorporated herein in their entirety by reference,which means it should be read and considered by the reader as part ofthis text. That the document, reference, patent application or patentcited in the text is not repeated in this text is merely for reasons ofconciseness. Reference to cited material or information contained in thetext should not be understood as a concession that the material orinformation was part of the common general knowledge or was known in anycountry.

Throughout the specification, unless the context demands otherwise, theterms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or‘comprising’, ‘includes’ or ‘including’ will be understood to imply theincludes of a stated integer or group of integers, but not the exclusionof any other integer or group of integers.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be discussed by way ofexample only with reference to the figures in which:

FIGS. 1a and b illustrate example HPLC-size exclusion chromatograms ofP. capsulatus polysaccharide showing purity and molecular weight,wherein (a) is an example chromatogram from the HPLC-size exclusionanalysis of polysaccharide using refractive index detection and thesample is above the resolution of the Biosep 4000 column (top standard670 kDa) and (b) is example chromatograms and contour plot from the sizeexclusion analysis of polysaccharide using photodiode array detectionwherein sample is above the resolution of the Biosep 4000 column used.Minimal absorbance at low wavelength only.

FIGS. 2a and b illustrate two example HPLC-size exclusion chromatogramsof P. capsulatus polysaccharide derivatives showing purity and molecularweight based on using refractive index detection, YMC120 Diol column andpeaks after 6.5 minutes are associated with the mobile phase buffer.Example batches indicate good reproducibility of process and 1→8 kDarange of LMW material.

FIGS. 3a, 3b, 3c and 3d illustrate example chromatograms from themonosaccharide analysis of P. capsulatus polysaccharides andpolysaccharide derivatives using methanolysis-TMS derivatisation GC-FIDmethod, wherein (a) is an example chromatogram of the mixedmonosaccharide standards used in the methanolysis—TMS GC-FID method withtabulated data below; (b) is an example chromatogram from themonosaccharide analysis of P. capsulatus polysaccharide showing the keymonosaccharide peaks, based on comparison with mixed standards andinternal standard ratio with tabulated data below; (c) is an examplechromatogram from the monosaccharide analysis of polysaccharidederivatives (large polysaccharide fragment 20-60 kDa), showing the keymonosaccharide peaks, based on comparison with mixed standards andinternal standard ratio with tabulated data below; (d) is an examplechromatogram from the monosaccharide analysis of polysaccharidederivatives (small polysaccharide fragment 2-10 kDa), showing the keymonosaccharide peaks, based on comparison with mixed standards andinternal standard ratio with tabulated data below.

FIG. 3e illustrates a table of example data from the monosaccharideanalysis of P. capsulatus polysaccharide and polysaccharide derivativescomparing the methanolysis—GC-FID method with a TFA-hydrolysis—HPAEC-PADmethod wherein repeat samples of the small polysaccharide derivatives(2-10 kDa) and the HMW native polysaccharide represent differentpreparations. Data shown as % monosaccharide composition. Both methodsshow the presence of similar monosaccharides although percentages vary.Example data from the TFA/HPAEC-PAD method shows the presence ofgalactose in all samples, although at a lower percentage than themethanolysis-TMS GC-FID method. This could be due to the stronger acidconditions (TFA) used for hydrolysis.

FIGS. 3f and g illustrate example paper chromatograms of TFA treatedhydrolysates of P. capsulatus polysaccharide and polysaccharidederivatives to determine monosaccharide composition wherein (f)illustrates example results from paper chromatography of P. capsulatuspolysaccharide hydrolysates from TFA-HPAEC monosaccharide analysismethod (data shown in FIG. 3e ) illustrating monosaccharide composition.Each marker is 25 μg. Solvent ethyl acetate:pyridine:water 8:2:1 Stain:aniline hydrogen-phthalate (where Xyl=xylose, Fuc=fucose, Ara=arabinose,Man=mannose, Glc=glucose, Gal=galactose, GalA=galacturonic acid,Rib=ribose, Rha=rhamnose, Samp=P. capsulatus polysaccharide sample). Themajor monosaccharides identified in the P. capsulatus polysaccharide andpolysaccharide derivatives, support the data shown in FIG. 3b-e . FIG.3g illustrates example results from paper chromatography of P.capsulatus hydrolysates from TFA—HPAEC monosaccharide analysis method(FIG. 3e ) illustrating monosaccharide composition. Each marker is 25μg. Solvent butanol:acetic acid:water 12:3:5 Stain: anilinehydrogen-phthalate (where Xyl=xylose, Fuc=fucose, Ara=arabinose,Man=mannose, Glc=glucose, Gal=galactose, GalA=galacturonic acid,Rib=ribose, Rha=rhamnose, Samp=P. capsulatus polysaccharide sample). Themajor monosaccharides identified in the P. capsulatus polysaccharide andpolysaccharide derivatives, support the data shown in FIG. 3b -f.

FIG. 4 illustrates example molecular weight estimation of P. capsulatuspolysaccharide using sedimentation equilibrium wherein an examplecalculation of approximate molecular weight of the P. capsulatus nativepolysaccharide using sedimentation equilibrium techniques is provided.Sample was run at 0.5 mg/ml in 0.1M NaCl. Sedimentation velocityexperiment was carried out to assess heterogeneity, before molecularweight analysis by sedimentation equilibrium. Data fit was achieved at arotor speed of 1400 rpm with 1 mm column. P. capsulatus nativepolysaccharide was calculated to have a molecular weight of 38.62 MegaDaltons.

FIG. 5 illustrates an example size exclusion chromatogram showing thepurification of P. capsulatus polysaccharide derivatives afterfree-radical depolymerisation. Size exclusion chromatographypurification was using (Superdex 30 resin) and desalting of P.capsulatus polysaccharide derivatives after depolymerisation. Absorbanceat 214 nm (light blue) (i), A280 (dark blue (ii)) (top panel) andconductivity (green) (iii) (lower panel) monitored. 3 ml fractionscollected from 30 mins onwards, with 3 ml delay between UV monitor andfraction collector. Two broad size ranges can be seen in the main peaksand these were collected:

43-61 minutes=large fragment derivatives (20-60 kDa),

61-81 minutes=small fragment derivatives (2-10 kDa).

Material eluting after 100 minutes (co-eluting with the salt peak—green(iii)) are monosaccharides.

FIG. 6 illustrates the effects of P. capsulatus polysaccharide, andderivatives of polysaccharide, on human neutrophil elastase activitywherein there is shown example data for the effects of P. capsulatuspolysaccharide, and polysaccharide derivatives on human neutrophilelastase activity (all samples at 0.1 mg/ml). Data illustrates the meanelastase activity±SD, % relative to control, from several batches ofproduct. Treatment with algal polysaccharide and derivatives results ina reduction of elastase activity.

FIG. 7 illustrates the effects of P. capsulatus polysaccharide, andderivatives of polysaccharide, on human neutrophil ROS productionwherein there is shown example data for the effects of P. capsulatuspolysaccharide, and polysaccharide derivatives on human neutrophilreactive oxygen species (ROS) production (all samples at 0.1 mg/ml).Data illustrates the mean ROS signal±SD, % relative to control, fromseveral batches of product. Treatment with algal polysaccharide andderivatives results in reduction of ROS.

FIG. 8 illustrates the effects of P. capsulatus polysaccharide, andderivatives of polysaccharide on BHK cell viability wherein there isprovided example data for the effects of P. capsulatus polysaccharideand polysaccharide derivatives on BHK cell viability (all samples at 0.1mg/ml). The data illustrates the mean cell viability±SD, % relative tocontrol, from several batches of product. Treatment with algalpolysaccharide and derivatives results in no effects on observed cellviability.

FIGS. 9a and 9b illustrates the effects of P. capsulatus polysaccharidederivatives on IL8 release or IL8 gene expression by primary humankeratinocytes after stimulation with 10 ng/ml of IL1beta or 10 ng/mlTNFalpha wherein (a) is Example data for the effects of P. capsulatuspolysaccharide and polysaccharide derivatives on IL8 release fromprimary human keratinocytes after stimulation with 10 ng/ml of IL1beta(all samples at 0.1 mg/ml). The data illustrates the % release±SD,relative to the IL1beta only control. Fucoidan and heparin treatmentsare run for comparison. Treatment with polysaccharides results in areduction in IL8 secretion from keratinocytes; (b) Example data for theeffects of P. capsulatus polysaccharide derivatives on IL8 geneexpression by primary human keratinocytes after stimulation with 10ng/ml of TNFalpha (all samples at 0.1 mg/ml). The data illustrates the %relative gene expression±SD, using TNFalpha only control as thecalibrator, and GAPDH as a housekeeping gene. Fucoidan treatment run forcomparison. Treatment with polysaccharide derivatives results in areduction in IL8 gene expression by keratinocytes.

FIG. 10 illustrates the effects of P. capsulatus polysaccharidederivatives on IL8 release from primary human keratinocytes whereinthere is provided example data for the effects of P. capsulatuspolysaccharide derivatives (2-10 kDa) on IL8 release from primary humankeratinocytes after stimulation with 10 ng/ml of TNFalpha, 20 ng/mlIL17A, or both in combination. The data illustrates the keratinocyte IL8release in pg/ml for control wells (no stimulation or cytokine only) andwells treated with small polysaccharide derivatives, fucoidan, or NF-κBinhibitor SC-514. Treatment with polysaccharide derivatives results in areduction in IL8 secretion from keratinocytes.

FIGS. 11a and 11b illustrate the dose-dependent effects of P. capsulatuspolysaccharide derivatives on IL8, IL17C and IL6 release by primaryhuman keratinocytes wherein (a) there is provided example data for theeffects of P. capsulatus polysaccharide derivatives (2-10 kDa) on IL8release from keratinocytes stimulated with 10 ng/ml TNFa & 50 ng/mlIL17A. The data indicates that the polysaccharide inhibits the releaseof IL8 in a dose-dependent fashion. It has greater potency at similardoses than the NF-κB inhibitor (SC-514), and the MAPK p38 inhibitor(SB203580). Low molecular weight (LMW) heparin is shown for comparison;(b) example data for the effects of P. capsulatus polysaccharidederivatives (2-10 kDa) on IL17C & IL6 release from keratinocytesstimulated with 10 ng/ml TNFa & 50 ng/ml IL17A & 10 microM histamine.The data indicates that the polysaccharide inhibits the release of IL17C& IL6 in a dose-dependent fashion. Low molecular weight (LMW) heparin isshown for comparison.

FIG. 12 illustrates the dose-dependent effects of P. capsulatuspolysaccharide derivatives on IFNgamma release by primary humanperipheral blood mononuclear cells wherein there is provided exampledata for the effects of P. capsulatus polysaccharides derivatives (2-10kDa) on release of interferon gamma (IFNgamma) from peripheral bloodmononuclear cells stimulated with 10 microg/ml phytohaemagglutinin and10 ng/ml IL1beta. Data indicates that polysaccharide inhibits IFNgammarelease in a dose-dependent fashion. This effect varies depending on thelength of blood cell culture. Low molecular weight (LMW) heparin isshown for comparison.

FIG. 13 illustrates the effects of P. capsulatus polysaccharidederivatives on primary human neutrophil chemotaxis wherein there isprovided example data for the effects of P. capsulatus polysaccharidederivatives on IL8 stimulated neutrophil chemotaxis (all samples at 0.1mg/ml). Relative luminescent units±SD indicates the number of migratedneutrophils (which are visualised by a luminescent signal) in eachtreatment, where IL8 only is the control. Treatment with polysaccharidederivatives results in a reduction in neutrophil chemotaxis.

FIG. 14 illustrates the dose-dependent effects of P. capsulatuspolysaccharide derivatives on THP-1 (monocyte) chemotaxis wherein thisis provided example data for the effects of P. capsulatus polysaccharidederivatives (2-10 kDa) on MCP-1 (monocyte chemoattractant protein)stimulated THP-1 (pro monocytic cell line) chemotaxis. Treatment withpolysaccharide results in a dose-dependent reduction of THP-1chemotaxis. There is some variation between preparations, with thelarger fragments showing greater inhibition (60-70%) although this couldbe a non-specific effect.

FIG. 15 illustrates the dose-dependent effect of P. capsulatuspolysaccharide derivatives on mouse skin inflammation wherein there isprovided example data for the effects of P. capsulatus polysaccharidederivatives (2-10 kDa) on imiquimod (IMQ) induced mouse skininflammation after 9 days of IMQ treatment and polysaccharide dosing.Inflammation is measured by scoring of histological parameters includingepidermal and keratinocyte proliferation, and leukocyte infiltration.The polysaccharide shows inhibition of skin inflammation in adose-dependent manner.

* denotes statistical significance by ANOVA with Dunnett's post hocanalysis.

FIG. 16a illustrates a summary table of characterising features of P.capsulatus polysaccharide and polysaccharide derivatives thereof of theinvention.

FIG. 16b illustrates the calculated sulphate content of P. capsulatuspolysaccharides based on two different methods of analysis: abiochemical plate assay for sulphate (Terho method), compared withICP-OES for sulphur (followed by a mathematical conversion to sulphate).The % sulphate detected is in good agreement between the two methods,confirming the high sulphate content of the polysaccharide.

EXAMPLES Example 1 Growth of Prasinococcus capsulatus Strain Cultures

P. capsulatus strain cultures were maintained in the laboratory at 50 or100 mL f/2 medium, with continuous light and at ambient (lab)temperatures (range throughout the year 18-28° C.). To set up the largerlaboratory cultures, the stock culture was initially inoculated into 250ml or 500 ml of f/2 medium and grown to log phase. 250 ml or 500 mlcultures were inoculated into 10 or 20 liter clear polycarbonate carboyswith a vent screw with PTFE air inlet and air exhaust filters,containing f/2 medium autoclaved 116° C. for 30 mins. Inorganicphosphate and trace metals were autoclaved singly and separately addedto the bulk of the medium after autoclaving.

Different amounts of CO₂/air mixes can be used, with different levels ofillumination and temperature. Specifically 10 and 20 L cultures weresparged vigorously with air, with continuous illumination, using whitefluorescent tubes at ambient (lab) temperatures: 18-28° C. deg rangethroughout year.

Alternatively many different pilot and large-scale culture systems couldbe used to grow P. capsulatus. Specifically a 200 liter pilot scalemicroalgal photobioreactor system (International patent WO2011/031161A1; Norwegian patent 320950) was used with f/2 growth medium, aerationwith air and 1-2% CO₂, and with continuous illumination (350micromol/m²/sec; 7-10 cm light path) between 18-28° C. temperaturerange.

Example 2 Harvesting Polysaccharide from Culture Medium

Polysaccharide can be obtained from culture medium by variouscentrifugation and filtration techniques. Specifically dense laboratorycultures (10-20 liters) were harvested and transferred to 4×600 mlcentrifugation pots of Heraeus Multifuge 3L-R centrifuge. Centrifugationwas at 4500 g for 2 hours per batch. The culture medium (supernatant)was transferred whilst the algal cells were pelleted in thecentrifugation pot. The supernatant was further clarified by vacuumfiltration (to remove residual cells), using a Whatman no. 3 filter.

This supernatant was then subjected to cross flow filtration using aPall Centramate with a 0.1 m² SkDa molecular weight cut off (MWCO)T-series membrane. Other MWCO's can be used, as long as below MW ofpolysaccharide. Sample was refiltered through a Whatman No. 3 filterpost-thawing, before being circulated through the membrane. Retentatewas re-circulated until concentrated to ×10 original volume. This wasthen rediluted to original volume and repeated, to ensure salt and mediacomponents were removed in the permeate. Conductivity was monitoredduring the process. The retenate sample was collected and spray driedusing a Buchi Mini Spray Dryer B-290 to provide dry polysaccharidepowder. Further MWCO using any appropriate technique can be used toisolated target polysaccharide, but specifically separation using 300kDa MWCO Vivaspin (Sartorius) by centrifugation was used to generate ahigh MW fraction.

Alternatively many different pilot and large-scale systems could be usedto harvest polysaccharide from the culture medium of large-scalecultures. Specifically a disc stack Westphalia centrifuge was used toseparate cultures from 200 liter photobioreactors running at 20-60liters/hour. The culture was pumped directly into the disc-stackcentrifuge from the microalgal photobioreactor, with the mediumcomponent being collected in an IBC.

The supernatant was then pre-filtered with a 5 micron filter (PURTREXPX05-9 7/8) and subjected to cross flow filtration using a Combi SystemM38-H-2.25-3 (Alfa Laval), with GR60P 25 kDa MWCO membranes. Sample wasconcentrated×10 and then 3 volumes of water (600 liters) were added todiafilter (removal of salts, medium components and low molecular weightmaterial). Conductivity was monitored throughout, and the retentatesample collected. The polysaccharide containing retentate can be furtherspray dried using many different large-scale drying systems.Specifically a Mobile Minor spray drying system (GEA ProcessEngineering) was used, with inlet temperature of 200° C., and outlettemperature of 90-99° C., by atomising 2.3-2.7 kg/hour and recoveringthe dried polysaccharide from the cyclone and bag filter.

Example 3 Harvesting Polysaccharide from Cell Pellets

The cell pellets resulting from the removal of media can also beprocessed to generate target polysaccharide. This could be carried outby hot water extraction techniques, enzymatic digest or various otherextraction protocols. Specifically cells were transferred to 50 mLsterile tubes and centrifuged for 2 h at 10,000 g. Remaining supernatantwas removed and cells stored frozen for later extraction and processing.To process, the cells can be lysed and product removed using standardextraction techniques. Specifically cells were freeze-thawed three timesprior to extraction. They were then mixed with an equal volume ofTris-HCl pH8, mixed with the enzyme alcalase 2.5 L DX (Novozyme) at aconcentration of 1 vol enzyme to 100 vols sample, and incubatedovernight at 60° C. with stirring. The sample was removed, spun at10,000 g to pellet cell debris and the supernatant removed. Thissupernatant was dialysed against water using 8 kDa MWCO membrane using10 volume batches of water with 4 changes over 36 hours. The desaltedsupernatant can be freeze or spray dried to generate a polysaccharidesample. Further MWCO using any appropriate technique can be used toisolated target polysaccharide, but specifically separation using 300kDa MWCO Vivaspin (Sartorius) by centrifugation was used to generate ahigh MW fraction.

Example 4 Depolymerisation and Oligosaccharide Purification

High molecular weight target polysaccharide can be depolymerised intosmaller fragments using techniques such as enzymatic digestion or acidhydrolysis. Specifically it can be depolymerised by introduction ofhydrogen peroxide into a hot polysaccharide solution, to generate freeradicals, which attack glycosidic bonds (Rota C et al. 2005 Free radicalgeneration during chemical depolymerization of heparin. Anal Biochem.344(2): 193-203. and Petit A C et al. 2006 Free-radical depolymerizationwith metallic catalysts of an exopolysaccharide produced by a bacteriumisolated from a deep-sea hydrothermal vent polychaete annelid.Carbohydrate Polymers 64: 597-602.). Key variables are the ratio ofhydrogen peroxide to polysaccharide, temperature and pH control. Solidpolysaccharide sample was added to water at approximately 2 mg/ml,dissolved and warmed to 60° C. in a water bath with stirring. Coppersalt solution was added to give a 0.01M concentration. Using a pHcontroller connected to a pump containing sodium hydroxide, the samplewas set to pH 7.5. At this point the reaction is started by pumpinghydrogen peroxide into the vessel at a constant flow rate, e.g. 0.5ml/min, with the pH controller set to maintain the pH at 7 by turningthe sodium hydroxide pump on when required. Once the reaction had beenrun for the desired period the pumps were stopped and pH was loweredusing 20% acetic acid (5 microL/ml of reaction), chelex 100 (Sigma) wasadded at 60 mg/ml of reaction and the reaction is mixed on a rotatingstirrer until clear. The whole reaction was removed from the chelex andstored at −20° C. Products were purified from the reaction by exchangeof any remaining copper ions with sodium ions using Q-sepharose (GE)anion exchange followed by desalting/separating by size exclusionchromatography by Superdex 30 (GE) using bench columns and BuchiSepacore system with detectors for A214, A280 and conductivity. TheQ-sepharose column was equilibrated in 50 mM Tris-HCl pH7.5, 50 mMsodium chloride mobile phase, followed by loading of thedepolymerisation reaction, and washing for a further 20-30 mins withmobile phase all at 10 ml/min. Then the bound polysaccharide was elutedwith 5M sodium chloride solution and collected. The eluate was added in5 ml batches to a size exclusion bench column Superdex30 at 1 ml/minwith water as mobile phase. Separation was carried out over 120 mins,with 3 ml fractions collected using a Pharmacia fraction collector.Fractions of different polysaccharide molecular weight ranges wereidentified, pooled, and freeze-dried. Specifically two size ranges werenormally collected representing 2-10 kDa and 20-60 kDa derivatives butother sizes may be collected. Stock solutions of derivatives wereinjected onto a size exclusion HPLC column (Biosep4000, YMC Diol300 orYMC Diol120) to confirm approximate molecular weight (see below).

Example 5 Determination of Approximate Molecular Weight

Polysaccharide and polysaccharide derivative molecular weight wasestimated by size exclusion chromatography using a Waters Alliance HPLC(2695) with Refractive index (Waters 2410) and Photodiode Array (210-380nm) detection (Waters 996). Either YMC300-Diol or Biosep4000 sizeexclusion columns were equilibrated at 30-37° C. in 0.2 micron filtered50 mM Tris-HCl pH7, 1 mM EDTA, and 0.9% NaCl mobile phase. Columns werecalibrated using dextran standards (Fluka: 12, 27, 50, 80, 270, 670kDa), by injecting 20 microL in mobile phase, running at 0.5 or 1 ml/min(YMC300 or Biosep4000) with 10 or 15 minute isocratic separations. Thestandard curve was generated using the formula Kay=(retentiontime−V0)/(Vt−V0), and plotting Kay versus molecular weight. Samples wereinjected at 20 microL of a 0.1 mg/ml solution in mobile phase and run asper standards. Data was manually integrated with Millennium Waterssoftware, with or without blank baseline subtraction. The retentiontimes of the sample were compared to those generated for the standardcurve to calculate approximate molecular weight using the formula above.

Further, the molecular weight of the native P. capsulatus polysaccharidecan be estimated by sedimentation equilibrium techniques. Specificallysedimentation equilibrium using a Beckman XL-I analytical centrifuge(AUC) equipped with scanning absorption optics was used. Samples wereresuspended at 0.5 mg/ml in 0.1M sodium chloride in water. Asedimentation velocity experiment was carried out to assess sampleconfirmation and heterogeneity, and assess required parameters forsubsequent sedimentation equilibrium. Sedimentation equilibrium wascarried out at rotor speed of 1400 rpm and 1 mm column.

Example 6 Determination of Approximate Sulphate Content

Various methods may be used to determine sulphate content of targetpolysaccharide and polysaccharide derivatives. Specifically sulphatedetermination was carried out based on a method by Terho T & Hartiala K(Method for the determination of sulphate content of glycosaminoglycans.Analytical Biochemistry (1971) 41 (2): 471-476). 25 μl of 1 mg/ml sampleor control (chondroitin Sigma C4384 or heparin Sigma H3393) in water wasplaced in a reaction vial. 1M HCl was added to give a finalconcentration of 0.5-1M HCl and the vials heated at 100° C. for 2 hours.The hydrolysed sample was rotary evaporated using a Speed Vac (JouanRC10/10 with RCT60 refrigerated trap) under vacuum at 60-65° C. untildry (usually 1-2 hours). The dried hydrolysate was dissolved in 250microL of water (0.1 mg/ml).

Standards were prepared from 1 mM sulphuric acid to give concentrationsin the assay of 0.048, 0.096, 0.192, 0.288, 0.384, 0.432, 0.48 μgsulphate. 100 microL of each sample, standard, control or blank (wateronly) were pipetted into an eppendorf, to which 400 microL of ethanolwas added and mixed thoroughly. 125 microL of this mix was added totriplicate wells of a 96 well assay plate, 50 μl BaCl₂ buffer (freshlyprepared 0.2M Acetic acid, 0.1 mM barium chloride, 1.6 mM sodiumhydrogen carbonate all in absolute ethanol) was added to each well,followed by 75 μl sodium rhodizonate solution (freshly prepared 0.05mg/ml, 1 mg/ml L-ascorbic acid in absolute ethanol). The plate wasshaken at medium speed for 30 secs, incubated in the dark for 10 minutesand shaken again. Colour intensity was measured in an absorbancemicroplate reader (BioTek Power Wave HT) at 520 nm using GenS software.Absorbance was calculated by subtracting the mean absorbance for eachsample, standard or control from the mean absorbance of the blank. Astandard curve was generated by plotting the blanked absorbance againstthe sulphate concentration for each sulphuric acid standard, and thesulphate content of samples and controls was interpolated. This value iscorrected for dilutions and volumes to give %sulphate=((MeanΔA520×40)/50)×100.

Sulphate determination was also carried out by inductively coupledplasma optical emission spectrometry (ICP-OES). 10 mg of polysaccharidewas resuspended in 1 ml water and then extracted in nitric acid:hydrochloric acid (aqua regia). Samples were nebulised and the aerosolproduced was transported to a plasma torch where excitation occurred.Characteristic atomic line spectra for sulphur were produced by aradio-frequency inductively coupled plasma. The spectra were dispersedby a grating spectrophotometer and intensities of the lines weremonitored by photomultiplier tubes. The photocurrents from thephotomultiplier tubes were processed and sulphur content calculatedbased on sulphur standards (multipoint calibration) and converted tosulphate value by mathematical formula.

Example 7 Determination of Monosaccharide Composition

Monosaccharide composition can be determined using a number of differentmethods. Specifically, monosaccharide composition was determined bymethanolysis and trimethylsilane (TMS) derivatisation followed bycomposition analysis using Shimadzu GC-2014 with flame ionisationdetection (GC-FID). Reaction vials were heat cleaned in a furnace ovenfor 6 hours at 450° C. 100 microg of sample (as a 10 mg/ml solution) wastransferred to a vial and 5 nmol of scyllo-inositol internal standardwas added to each sample. A vial containing 5 nmol of eachmonosaccharide standard was also set up containing scyllo-inositol(18132 Sigma), arabinose (A3131 Sigma) xylose (X-1500 Sigma), mannose(M6020 Sigma), fucose (F2252 Sigma), rhamnose (R3875 Sigma), galactose(G0750 Sigma), glucose, glucosamine (G4875), galactosamine (G0500),glucuronic acid (G5269), galacturonic (48280 Fluka), sialic acid (allprepared as 100 mM stock solutions). All vials were dried in a speed-vac(Jouan RC10/10 with RCT60 refrigerated trap) under vacuum at 60-65° C.until dry (usually 1-2 hours). 40 microL of neat methanol was added, thesamples dried again as above and then resuspended in 100 microL of 0.5Mmethanolic HCl. The vials were heated at 85° C. in a heat bloc for 4hours. After cooling 20 microL neat pyridine was added to neutralise theHCl and 20 microL neat acetic anhydride was then added to re-N-acetylateany free primary amines (for 30 minutes at room temperature). The vialswere then dried again (speed vac as above), 40 microL of neat methanolwas added to wash, the vials were re-dried (10-30 mins speed vac asabove). 40 microL of neat TMS reagent was then added and mixedthoroughly to resuspend the sample. The vials were sealed and left forat least 10 minutes before injecting 1 microL onto a Shimadzu GC-2014with flame ionisation detection (300° C. splitless injection). Thecolumn was ZB5-ms, 30 m×0.25 mm i.d.×0.25 μm film thickness. Thechromatograms generated were analysed. The area cut off was manuallyadjusted for each sample until 20-30 peaks were identified. Peak areasand retention times were correlated with the monosaccharide standards.

Each peak was calculated:

ratio=peak area/internal standard peak area;

standard ratio=standard area/internal standard area for each standard;

nmoles=(5 nmoles/standard ratio)×sample ratio;

% of each monosaccharide present in the original sample=nmoles/totalnmoles×100.

Monosaccharide composition of P. capsulatus polysaccharide was alsodetermined by trifluoroacetic acid (TFA) hydrolysis followed by highperformance anion exchange chromatography with pulsed amperometricdetection (HPAEC-PAD).

Samples of P. capsulatus polysaccharide and derivatives were resuspendedat approximately 10 mg/ml in 2M TFA. They were incubated at 120° C. for1 hour in a heating block, with vigorous shaking after 30 mins. Theywere then centrifuged and the supernatant recovered. The pellet waswashed with 0.5 ml of water, re-centrifuged and the wash pooled with theoriginal supernatant. The recovered solution was dried in vacuo toremove the TFA and the dry residue was resuspended in 1 ml of water. A1/10th dilution of the sample was injected onto a Dionex PA1 columnalong with a dilution series of standard monosaccharides, disaccharidesand uronic acids. (This mix contained fucose, rhamnose, arabinose,galactose, glucose, xylose, mannose, ribose, cellobiose, maltose,galacturonic acid, glucuronic acid.). Samples were separated by gradientand resolved with PAD. Recovery of monosaccharides was calculated bycomparison to standards.

10 microL of the TFA hydrolysates (nominally 100-160 microg ofpolysaccharide) were also loaded onto 2 sheets of Whatman number 20paper. Monosaccharide standards mix—ribose, rhamnose, xylose, fucose,arabinose, mannose, glucose, galactose, galacturonic acid—was alsoloaded. Paper chromatography was carried out by running one sheet inbutanol/acetic acid/water 12:3:5 mobile phase for 30 hours, and theother in ethyl acetate/pyridine/water 8:2:1 mobile phase for 3 days withseparation by charge. Sheets were stained with anilinehydrogen-phthalate to visualise monosaccharides.

Example 8 Determination of Cytotoxicity

Various different cell-based screening assays can be used to determinethe cytotoxicity of the target material. Specifically cytotoxicity isexamined by measuring the effects of the polysaccharide andpolysaccharide derivatives on the metabolic activity of a BHK cell line(hamster kidney fibroblast ECACC 85011433). 90% confluent BHK cells areharvested and plated in a 96-well white microplate at 1×10⁴ cell/well in100 microL freshly prepared culture media (Glasgow Minimum EssentialMedium (GMEM), 10% Foetal Calf Serum, 5% Tryptose Phosphate Broth, 2 mML-Glutamine). They are left for 1 hour at 37° C. 5% CO₂ to allow>80%adhesion to the well. 11 microL of 1 mg/ml polysaccharide sample inHanks Balanced Salt Solution (HBSS), HBSS only control, fucoidan (1mg/ml in HBSS) control, and doxorubicin (10 microg/ml, 1 microg/ml inHBSS) controls are added to triplicate wells and the plate incubated for16-18 hours at 37° C. 5% CO₂. The plate is allowed to come to roomtemperature for 30 minutes before additions of 100 microL Cell titreglow reagent (Promega). Plate is mixed for 2 minutes on a plate shakerand then incubated for 10 minutes at room temperature. The resultingluminescence for each well is measured on plate reader (BioTek, Synergy3) using GenS software. Mean luminescence for each sample or control iscalculated. The HBSS control well is designated as 100% metabolicactivity and sample luminescent values are calculated against this %activity=(test well/control well)*100. The fucoidan and doxorubicincontrols should be within established values.

Example 9 Effects on Neutrophil Elastase Activity

Different protocols are possible for the measurement of the effect ofpolysaccharide on neutrophil elastase enzyme activity. Specificallyelastase activity was measured by incubation of polysaccharide withstimulated freshly isolated human neutrophils followed by reaction ofreleased enzyme with a labelled substrate and colourimetric measurementon a plate reader. Freshly isolated human neutrophils were resuspendedin HBSS (without Ca and Mg) and cells counted on a haemocytometer. Thecells were centrifuged and resuspended in HBSS to give a concentrationof 2.5×10⁶ cells/ml. 22 microL of sample, controls or HBSS were added toa microtube followed by: 25 microL of cytochalasin B (at 40 mg/ml inHBSS to give a final concentration 5 mg/ml); 25 microL of TNF a (at 80ng/ml in HBSS to give a final concentration of 10 ng/ml, with 25 microLHBSS used in place of TNF a for a non-stimulated control); 150 microL ofneutrophil suspension (or for a media only control group add 150 microLof HBSS in place of cells). Contents were gently mixed and the tubesincubated at 37° C. for 30 minutes. After incubation 25 microL of fMLP(at 1 microg/ml in HBSS to give a final concentration of 100 ng/ml) wasadded, or HBSS to the non-stimulated control group. Tubes were incubatedfor a further 45 minutes at 37° C. Tubes were centrifuged at 5000 rpmfor 5 minutes on a Heraeus Biofuge to pellet the cells and 25 microL ofthe supernatant is transferred into triplicate wells of a 96 well blackmicroplate. 150 microL of Tris HCl pH 7.5 and 20 microL of neutrophilelastase substrate 1 (0.5 mg/ml in Tris-HCl pH 7.5) were added to eachwell, except for a blank well which contains no substrate. The plate wastransferred to a prewarmed (37° C.) plate reader (BioTek Powerwave HT)and readings are taken at 405 nm every 5 minutes for 1 hour using GenSsoftware. Vmax was calculated over 4 data points between 10 minutes and1 hour. Mean Vmax was calculated for each sample, control or blank. Thecontrol well (stimulated cells with substrate, but no test samples) Vmaxwas designated as 100% and samples and controls are calculated againstthis to generate % elastase activity: % activity=(test well Vmax/controlwell Vmax)*100.

Example 10 Effects on Keratinocyte Cytokine Release and Gene Expression

The effects of polysaccharides and polysaccharide derivatives onkeratinocyte cells may be assessed using a range of different cell linesor primary cells, in various different growth media with or withoutpro-inflammatory stimulus. The resulting cytokine release can beassessed by different methods such as multiplex arrays or ELISA's.Specifically primary keratinocytes (Promocell C12003) were grown in fullkeratinocyte growth media with calcium and supplements (PromocellC20011) at 37° C., 5% CO₂ until 70-90% confluent. They were harvested bytrypsinisation, washed and seeded in the wells of a 24 well plate tissueculture plate at 30,000 cells per well. Cells were grown until ˜80-90%confluent (˜56 hours) and the media was then changed to basal media(Promocell C20211) with 0.5 mM calcium, for a further 16-18 hours.Samples (1 mg/ml in HBSS), controls (fucoidan 1 mg/ml in HBSS) or blanks(HBSS vehicle only) were then added to the wells (×10 dilution) for 6-8hours before addition of pro-inflammatory stimulus, or 1-2 hours in thecase of SC514 and SB203580 (NfkB and MAPK p38 inhibitor respectively).Pro-inflammatory stimulus was either 10 ng/ml TNFalpha or 10 ng/mlIL1beta or 20 ng/ml IL17A, or both 10 ng/mlTNFalpha and 20 ng/mlIL17 incombination. Other stimuli were 10 ng/ml TNFalpha and 50 mg/mlIL17A incombination, or 10 ng/ml TNFalpha, 50 mg/mlIL17A and 10 microM histaminein combination. Cells were incubated for a further 16-18 hours at whichpoint the supernatant was collected and stored at −80° C. Duringcollection the supernatant was replaced with PBS to wash the cells andthen this was replaced with 350 microL RNAeasy lysis buffer (Qiagen).Buffer, lysed cells and cell content were transferred to tubes forstorage at −80° C.

The collected supernatant was analysed for human IL8, IL6 or IL17Ccontent by ELISA (Peprotech for IL8 and IL6; R and D Systems for IL17C).The assay was read on a microplate reader (BioTek PowerWave HT usingGenS software) at A450-630 nm, and quantification was made by readingoff the standard curve. The concentration of cytokine in theunstimulated control was subtracted from the concentrations in the testwells and stimulated control well. The stimulated control well wasdesignated as 100% secretion of IL8 and samples were calculated againstthis % secretion=(corrected test well pg/ml/corrected control wellpg/ml)*100.

RNA was extracted from the cell lysate using a Qiagen RNAeasy Plus Kit.The resulting RNA was quantified on a spectrophotometer to checkconcentration range. cDNA was generated by using Qiagen QuantitectReverse Transcription kit (starting volume of RNA is 4 microL) withgenomic DNA removal by enzymatic digestion, prior to the RT step. cDNAwas add to a PCR reaction (1 or 2 microL) containing primers for eitherIL8 (Qiagen) or GAPDH (Qiagen) as the housekeeping gene, and QiagenQuantiFast SYBR PCR master mix (25 microL reaction volume). PCR wascarried out on an Mx3005P Real-time PCR machine (Stratagene) usingstandard settings (60° C. annealing and extension, 40 cycles). Cyclethreshold (Ct) values were obtained for test and control samples usingMxPro software. Relative expression of target genes was assessed bycomparison of target gene and housekeeping gene (comparative expressionby ΔΔCT method, with target and housekeeping gene PCR showing comparableamplification efficiencies), and with the stimulated control well cDNAdesignated as the calibrator sample (100% gene expression).

Example 11 Effects on Cytokine Release from Stimulated Human PeripheralBlood Mononuclear Cells (PBMC's)

The effects of substances on the release of cytokines from PBMC's can bemeasured using many different cell formats, stimuli and durations.Specifically the effects of P. capsulatus polysaccharide derivativeswere measured by incubation with isolated PBMC's. PBMC's were isolatedfrom fresh blood by histopaque (Sigma) gradient centrifugation, washedin modified HBSS medium (PAA), and resuspended in complete RPMI 1640medium (PAA). 200,000 PBMC's were added to a 96 v-well polypropyleneplate, along with media only controls. Polysaccharides at selectedconcentrations, along with controls SC514 and SB203580 (NfkB and MAPKp38 inhibitor respectively) were added to cells and incubated for 1 hourat 37° C., 5% CO₂. Phytohaemagglutinin (PHA) (10 microg/ml) and IL1beta(10 ng/ml) were added to each well except for unstimulated controls andplates were incubated at 37° C., 5% CO₂ for 2 or 3 days. To collectmedia, plates were centrifuged at 1000 rpm and the supernatanttransferred to a further plate for storage at −80° C., prior to cytokineanalysis. The collected supernatant was analysed for interferon gamma(IFNgamma) content by ELISA (Peprotech). The assay was read on amicroplate reader (BioTek PowerWave HT using GenS software) at 450-630nm and quantification was carried out by reading off the standard curve.The concentration of cytokine in the unstimulated control was subtractedfrom the concentrations in the test wells and stimulated control well.The stimulated control well was designated as 100% secretion of IFNgammaand samples were calculated against this control=(corrected test wellpg/ml/corrected control well pg/ml)*100

Example 12 Effects on Oxidative Burst from Neutrophils

There are numerous protocols to measure the production of reactiveoxygen species from immune cells, using different cells, stimuli andsubstrates. Specifically inhibition of the oxidative burst bypolysaccharide and polysaccharide derivatives was measured using humanneutrophils, which were stained with the reagent DCFH-DA. Freshlyisolated human neutrophils were resuspended in HBSS (without Ca and Mg)and cells counted on a haemocytometer. Cells were resuspended at 1×10⁶in HBSS, mixed with an equal volume of DCFH-DA at 40 microM in HBSS andincubated for 30 mins at 37° C., 5% CO₂. 100 microL of stained cellswere added to each well of a black 96 well microplate, apart fromtriplicate wells of a blank (HBSS only) and unstained cells control. 20microL of 1 mg/ml of samples, HBSS or controls (diphenyleneiodiumchloride (DPI) 1 microM concentration in HBSS) were added to triplicatewells containing stained cells. Cells were stimulated to produce ROS bythe addition of 50 microL of PMA (4 nM in HBSS), except for nostimulation control wells. Fluorescence generated by the oxidation ofDCFH-DA by ROS was measured on a fluorescent plate reader (BiotekSynergy 3) at 37° C., 485/528 nm kinetic read every 10 minutes for 2.5hours. Mean fluorescence is calculated, and blanked. Mean fluorescentdata for the PMA stimulated cells was designated as 100% response andsamples and controls were evaluated against this: % oxidativeburst=(sample fluorescence/PMA stimulated cells fluorescence)*100.

Example 13 Effects on Blood Cell Chemotaxis

A chemotaxis assay can be carried out using different types of immunecell, with different chemotactic agents. Specifically the effects ofpolysaccharide and polysaccharide derivatives on the chemotaxis ofneutrophils were measured. Human neutrophils are isolated from freshblood using Histopaque. 90 microL of freshly isolated neutrophils at2.5×10⁶/ml in HBSS including BSA 0.1%, 25 mM HEPES (Sigma), are mixedwith 10 microL of test compound (at selected concentration eg. 2-50microM) or vehicle controls, in each well of a polypropylene 96 wellplate (Greiner) and pre-incubated for 30 minutes. While cells and testcompounds are pre-incubating the lower chamber of a chemotaxis 96 wellplate (3 micron mesh) (Corning) is prepared. IL8 (Sigma) is made up inHBSS including BSA 0.1%, 25 mM HEPES, at required dose (eg. 0.37 ng/mlto 10 ng/ml final concentration). 235 microL is added per lower assaychamber. For negative controls 235 microL of HBSS (with Ca/Mg, BSA 0.1%,25 mM HEPES) is used in place of IL8. The lower assay chamber is thenincubated at 37° C. CO2 5% for 30-60 mins to pre-equilibrate media. Theupper wells are then carefully transferred to the lower and 75 microL ofneutrophils from each well of the polypropylene pre-incubation plate isadded to a well in the upper chamber of the chemotaxis plate. The wholeplate is incubated at 37° C. CO2 5% for 30 min.

The assay plate is removed from the incubator. The upper well contentsare discarded and the upper wells are transferred to a white 96-wellplate containing Accutase enzyme (Sigma) at 180 microL per well at roomtemperature. The plate is placed on a plate shaker for 5 mins at roomtemperature. The upper chamber is discarded and the number of cellspresent in the white plate is measured using Cell Titer Glow reagent(Promega) according to the manufacturers instructions (see also example8). 100 microL CellTiter-Glo reagent is added per well of the 96-wellluminescence plate containing the mesh cells and Accutase and mixed on aplate shaker for 2 min RT then incubated for 10 min RT. The luminescencesignal is measured on a Synergy 2 plate reader using Gen 5 software.Data is blanked against the media plus cells only control and the %migration calculated by comparison to the IL8 only chemotaxis 100%control.

Further, effects of P. capsulatus polysaccharide derivatives on thechemotaxis of monocytes, using THP-1 human pro-monocytic cell line (HPA88081201) were also assessed. Polysaccharide derivatives (triplicatewells) at selected concentrations were mixed with THP-1 cells at2×10⁶/ml in RPMI 1640 medium (PAA) with 25 mM HEPES, 2 mM Glutamine and0.1% BSA (Sigma) in a 96 v-well polypropylene (PP) plate (Greiner). Acells only control was also set up in triplicate. The cells andpolysaccharides were incubated at 37° C. 5% CO2 for 30 minutes. 235microL of 10 ng/ml of monocyte chemoattractant protein-1 (MCP-1)(Peprotech) in the same medium was added to the lower chamber of a 96well 5 micron mesh chemotaxis plate (Corning), using assay medium as anegative control. The upper plate was refitted and the plate incubatedat 37° C. 5% CO2 for 30 minutes to pre-equilibrate media. The assay wascarried out by transferring 75 microL of THP-1 cells (˜150,000 cells)and test samples from the PP plate into the upper chamber of the MCP-1containing chemotaxis plate. Care was taken to ensure cells were fullysuspended and mixed well before transfer. The plate was incubated at 37°C. 5% CO2 for 120 minutes. Chemotaxis was measured by removing mediafrom the upper chamber, transferring the membranes to a plate containing180 microL of Accutase enzyme (Sigma), shaking for 5 minutes anddiscarding membranes. 100 microL CellTiter-Glo reagent (Promega—as forneutrophil chemotaxis) was added to the lower well of the assay plateand to the Accutase containing plate. They were shaken for 2 minutes,incubated for 10 minutes and then 200 microL of the well contents wastransferred to a white 96-well plate and the luminescence measured on aSynergy 2 plate reader (Biotek) using GenS software. Data was blankedusing the media and cells only control, values were pooled from thelower chamber and accutase samples, and % chemotaxis was calculated bycomparison to the cells only MCP-1 control wells (100% chemotaxis).

Example 14 Effects on Skin Inflammation in Imiquimod (IMQ) TreatedBALB/c Mice

There are numerous protocols to assess the effects of test substances onskin inflammation in mouse models, using different types of mice,genetic inducers and external stimuli. Specifically the effects of P.capsulatus polysaccharide derivatives on skin inflammation were assessedusing an IMQ-induced BALB/c mouse model. Test groups included a naïveplus vehicle group, an IMQ only group, an IMQ plus vehicle group, apolysaccharide plus vehicle group at 3 different concentrations (1, 0.1,0.01%) and a cyclophosphamide control (10 mg/kg in 0.5% CMC), with 8mice per group. Polysaccharides were dissolved in an aqueous gelcontaining a polyacrylate sodium salt, glycerol, paraben andimidazolidinyl urea (=vehicle). 500 microL of test gels were applieddaily to the shaved backs of mice, 4 hours prior to the application of50 mg of IMQ cream (5%). Vaseline was used in the case of naïve micetest group and the cyclophosphamide was dosed orally once a day.Observations of skin appearance (scaling, folding, erythema) were madeeach day to provide a disease activity index. Dosing was repeated for 4days, after which 4 mice from each group were sampled, and then untilDay 9 of the experiment when all remaining mice were sampled. Skinsamples from all mice were fixed in formalin, embedded, sectioned andstained with haematoxylin and eosin. Slides were scored for epitheliahyperplasia, keratinocyte proliferation (hyperkeratosis), leukocyteinfiltration and increased vascularisation. Scores were plotted againsttest agents to determine the effects of polysaccharide treatmentcompared to IMQ plus vehicle controls.

Although the invention has been particularly shown and described withreference to particular examples, it will be understood by those skilledin the art that various changes in the form and details may be madetherein without departing from the scope of the present invention.

The invention claimed is:
 1. An oligosaccharide derivative of a gelforming polysaccharide, wherein the polysaccharide is obtainable frommicroalgal cells from the order Prasinococcus capsulatus wherein the gelforming polysaccharide is a sulfated heteropolymer of molecular weightof at least 670 kDa comprising glucose, galactose, arabinose and uronicacid units, wherein said derivative has a molecular weight in the rangeof about 2 kDa to 60 kDa.
 2. A derivative as claimed in claim 1 whereinthe derivative has a molecular weight in the range 2 to 10 kDa.
 3. Aderivative as claimed in claim 2 wherein the derivative comprises byweight about 30 to 40% Glucose 30 to 40% Galactose 8 to 14% Arabinose 7to 11% Uronic acid and 1 to 10% of other sugar units.
 4. A derivative asclaimed in claim 1 wherein the derivative has a molecular weight in therange 20 to 60 kDa.
 5. A derivative as claimed in claim 4 wherein thederivative comprises by weight about 25 to 28% Glucose 35 to 55%Galactose 8 to 17% Arabinose 4-6% Uronic acids and 1 to 5% of othersugar units.
 6. A derivative as claimed in claim 1 wherein thederivative inhibits neutrophil elastase release by about 70 to 90%.
 7. Aderivative as claimed in claim 1 wherein the derivative inhibitsneutrophil reactive oxygen species production by about 30-40% relativeto neutrophils to which derivative has not been provided.
 8. Aderivative as claimed in claim 1 wherein the derivative inhibits humankeratinocyte IL-8 gene expression and IL8 release by about 70 to 100%relative to keratinocyte to which derivative has not been provided.
 9. Acomposition comprising a derivative, as claimed in claim
 1. 10. Acosmetic preparation comprising a derivative as claimed in claim
 1. 11.A nutritional supplement comprising a derivative as claimed in claim 1.12. A method for prophylaxis or treatment of an immune system disordercomprising administering the derivative of claim 1 to an animal in needthereof.
 13. The method of claim 12 wherein said immune system disorderis an inflammatory skin condition selected from the group consisting ofeczema, psoriasis and atopic dermatitis.
 14. The method of claim 12wherein said immune system disorder is an inflammatory condition of thegut selected from the group consisting of irritable bowel syndrome,Crohn's disease and ulcerative colitis.
 15. The method of claim 12wherein said immune system disorder is an inflammatory condition of therespiratory system selected from the group consisting of asthma, cysticfibrosis, emphysema, chronic obstructive pulmonary disorder, acuterespiratory distress syndrome, and allergic rhinitis.